Fig. 2.

Assessment of disease phenotypes in dHMNX patient-derived iPSCs. (A) Staining of iPSC clones shows decreased levels of ATP7A within the trans-Golgi (TGN46) in the patient cells. The boxed areas are enlarged and the DAPI channel removed to highlight increased levels of ATP7A in control iPSCs. (B) Quantification of the ATP7A mean fluorescence within the region of interest (ROI; defined by staining the cells with anti-TGN46 antibody) in patient and control iPSC lines. Violin plot shows full distribution of all data points acquired (n>1000 ROIs) and P-values were obtained by ANOVA followed by Tukey's post hoc test (****P<0.0001). (C) Levels of ATP7A in protein lysates determined by western blot analysis. Data in bar graphs are represented as mean±s.e.m. and P-values were obtained from a two-tailed Student's t-test (*P<0.05). (D) Cu content of iPSC pellets was measured in triplicate by ICP-MS following incubation of iPSCs with 0.5 µM and 100 µM CuCl₂ for 6 h. Data are expressed as the mean±s.e.m. (E) Cu-induced toxicity determined by CCK-8 assay (n=4) after culturing iPSCs in a range of CuCl₂ concentrations, from 0 to 200 μM, for 6 h.